The reaction of hemoglobin (Hb) with dinitrobenzenes (DNBs) was studied to develop a molecular-level understanding of such reactions that will enhance the development of toxicokinetic models that employ Hb adducts as biomarkers for exposure. Methemoglobin (metHb) is formed during the reaction and UV/VIS spectroscopy was used to follow the reaction of DNB isomers with deoxy-(dxHb), oxy-(HbO2) and carboncarboxy-(HbCO) hemoglobin. HPLC chromatography of dxHb treated with radiolabelled DNB was employed to detect possible adduct formation. Deconvolution of the spectra and the presence of well-defined isobestic points imply that DNB induces a direct conversion of dxHb to metHb, but little or no conversion occurs for either HbCO or HbO2. This implies that the reaction of DNB with Hb may require direct access to the heme and/or that the reaction is initiated by oxidation of the heme, which occurs more readily in the deoxy state. Labelled DNB formed no detectable covalent Hb adducts in the presence of dxHb, providing evidence that metHb formation is not linked to adduct formation.